Transrenal Mycobacterium tuberculosis DNA in pulmonary tuberculosis patients during the first 14 days of treatment

ABSTRACT We assessed the performance of a novel real-time PCR-based transrenal DNA (trDNA) assay for the specific detection of Mycobacterium tuberculosis as a candidate marker of the early anti-tuberculosis therapy response. The study was performed on 288 urine samples from 72 tuberculosis patients collected at baseline and days 3, 7, and 14 of treatment with amoxicillin-clavulanic acid alone or in combination with meropenem, ertapenem, optimized-dose rifampicin, or standard treatment control in South Africa. trDNA was detected in one-third of the samples. The highest proportion of positive PCR results (cycle threshold < 36) was observed on days 3 and 7, reflecting the point in time when maximum bacterial killing and disintegration are expected. When analyzed by study arms, the trend was observed in groups treated with active antibiotics affecting cell wall integrity (meropenem, control) but not in inactive drugs (ertapenem, amoxicillin/clavulanic acid alone) or active drugs not affecting the cell wall (rifampicin). Overall, however, the trDNA assay did not correlate well with sputum culture-based decline of viable bacteria. This is possibly due to trDNA reflecting the killing of both culturable and non-culturable bacteria and should be explored further. IMPORTANCE This study presents the results of the evaluation of a novel method for the detection of Mycobacterium tuberculosis, the causative agent of tuberculosis, in urine. Detecting parts of the mycobacteria in urine is of particular interest as it allows us to use a sample that is easy to obtain and that does not require uncomfortable procedures or safety precautions like obtaining sputum for culture, which is the most commonly used sample in the diagnosis of tuberculosis. In certain groups of individuals who cannot produce sputum, for example, children, non-sputum-based methods have particular importance. We found that the method tested was able to detect bacterial killing by active antibiotics that disrupt the cell wall and lead to fragmentation of bacteria. However, the assay can't detect inactive bacteria or bacteria that are active with an intact cell wall.

The manuscript of Kontsevaya et al. addresses the importance of biological tool, transrenal Mycobacterium tuberculosis DNA detection in urine sample of patients through RT-PCR.The authors demonstrate that this non-invasive technique could be good marker for detection of Mtb after early TB drugs treatment.Authors also mentioned that this method is not well correlated with sputum-based method.The manuscript is interesting, and presents a new methods for detection of Mtb.I have some major and minor comments on the manuscript: Major comment: 1. Sensitivity of this technique is attributed to action of cell wall targeting antibiotics.Will be interesting to see how well this technique works with urine samples from patients undergoing isoniazid (first line TB drug) and other cell wall targeting antibiotics therapy? 2. Is this method applicable for detection of bacterial load if sample is lated at late time points as after 2 weeks?3.In this manuscript authors did not discuss Mtb severity levels of the patients.Minor comment: Line 89: Is total number of samples 288 or 72 used in study?
Reviewer #2 (Comments for the Author): In this study the authors applied to detection of trDNA to urine samples to assess the potential of trDNA to detect bacterial killing in an EBA study.Although trDNA detection is too insensitive to be used as a diagnostic its use to follow bacterial killing in EBA studies would be interesting.The authors do indeed observe a moderate increase on the proportion of positive samples during treatment (Ct<36) but this peak does not correlate with the peak in EBA observed by culture.The authors suggest this may be due to killing of a different bacterial population which is indeed a possibility This is an interesting study and deserves to be reported.I have a couple of comments questions: What about the actual Ct values when was the lowest Ct value observed?In lines 168-68 the authors state they were not able to compare Ct values.How consistent were the Ct values between individual patients, what was the range of Ct values obtained for all patinets?Would it be possible to normalise to the day 1 Ct value (if positive) of patients with positive results?Or were the positive results randomly distributed between the patients, i.e. many / most patients only positive at one time point?Or to rephrase how many patients positive at time point one were positive/not positive at later time points?Would it be possible at least to present this information i.e. the samples positive at each time point (and the Ct value) for each patient may be in a supplementary file.

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The manuscript of Kontsevaya et al. addresses the importance of biological tool, transrenal Mycobacterium tuberculosis DNA detection in urine sample of patients through RT-PCR.The authors demonstrate that this non-invasive technique could be good marker for detection of Mtb after early TB drugs treatment.Authors also mentioned that this method is not well correlated with sputum-based method.The manuscript is interesting, and presents a new methods for detection of Mtb.I have some major and minor comments on the manuscript: Major comment: 1. Sensitivity of this technique is attributed to action of cell wall targeting antibiotics.Will be interesting to see how well this technique works with urine samples from patients undergoing isoniazid (first line TB drug) and other cell wall targeting antibiotics therapy? 2. Is this method applicable for detection of bacterial load if sample is lated at late time points as after 2 weeks?3.In this manuscript authors did not discuss Mtb severity levels of the patients.Please find a point-by-point response to the comments below.

Reviewer #1
Comment #1 by Reviewer #1: The manuscript of Kontsevaya et al. addresses the importance of biological tool, transrenal Mycobacterium tuberculosis DNA detection in urine sample of patients through RT-PCR.The authors demonstrate that this non-invasive technique could be good marker for detection of Mtb after early TB drugs treatment.Authors also mentioned that this method is not well correlated with sputum-based method.The manuscript is interesting, and presents a new methods for detection of Mtb.
I have some major and minor comments on the manuscript:

Authors´ response to the comment #1 of Reviewer #1: Comment #4 by Reviewer #2:
In this manuscript authors did not discuss Mtb severity levels of the patients.

Authors´ response to comment #4 of Reviewer #2:
We agree with the Reviewer that this aspect has not been discussed in the manuscript.The study participants were enrolled in an Early Bactericidal Activity study of drugs and combination with a strict set of inclusion and exclusion criteria.Participants with newly diagnosed, rifampicin susceptible pulmonary TB with at least 1+ smear microscopy, without HIV co-infection, diabetes, history of TB or signs of extrathoracic TB, among other criteria, were included.These participants are informally considered "healthy" TB patients and in terms of clinical well-being are quite similar.However, burden of disease can vary substantially from 1+ to 3+ and approximately 75% of the participants had cavitary lung disease which will be reported in the main paper currently in draft.trDNA positivity rate was compared to the time to positivity (TTP) of two sputum samples taken on the same day which is the indicator of bacterial load detected at a particular time point.

Comment #5 by Reviewer #2:
Minor comment: Line 89: Is total number of samples 288 or 72 used in study?

Authors´ response to comment #5 of Reviewer #2:
The study was conducted on 288 samples collected from 72 study participants.Four urine samples were collected from each participant during the 2-week treatment: on Day 1, 3, 7, and 14.This resulted in 288 urine samples in total.

General comment of Reviewer #2:
In this study the authors applied to detection of trDNA to urine samples to assess the potential of trDNA to detect bacterial killing in an EBA study.Although trDNA detection is too insensitive to be used as a diagnostic its use to follow bacterial killing in EBA studies would be interesting.The authors do indeed observe a moderate increase on the proportion of positive samples during treatment (Ct<36) but this peak does not correlate with the peak in EBA observed by culture.The authors suggest this may be due to killing of a different bacterial population which is indeed a possibility This is an interesting study and deserves to be reported.

Authors´ response to the general comment of Reviewer #2:
We thank the Reviewer for the high evaluation of our work.Authors´ response to comment #1 of Reviewer #2:

Comment #1 by
We thank the Reviewer for this interesting comment.In the study, Ct values ranged from 31 to 40 (the upper limit of measurement of the qPCR system used in the study).Each sample was tested in triplicate and the Ct values from three reactions were usually similar which points to the reproducibility of the method.It was not possible to analyse the consistency of Ct values between individual patients as the positivity rate was low but it seems that the results were not very consistent.Further optimisation of the assay is needed to improve the sensitivity and be able to conduct more a detailed analysis.

Comment #2 by Reviewer #2:
Would it be possible to normalise to the day 1 Ct value (if positive) of patients with positive results?Or were the positive results randomly distributed between the patients, i.e. many / most patients only positive at one time point?Or to rephrase how many patients positive at time point one were positive/not positive at later time points?Would it be possible at least to present this information i.e. the samples positive at each time point (and the Ct value) for each patient may be in a supplementary file.

Authors´ response to comment #2 of Reviewer #2:
We thank the Reviewer for this suggestion.The table with all results would be too long so we added Table S4 that only includes 20 study participants who had a positive PCR result on day 1.It can be seen that in most of the cases, if the sample collected on day 1 was PCR-positive samples on later time points will also be positive.Interestingly, in the majority of these cases, two sputum samples collected on the same day are also positive with a time to positivity of less than 7 days.We added these observations in Lines 153-159.Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.You will be notified when your proofs are ready to be viewed.
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Sincerely,
Po-Yu Liu Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org Is total number of samples 288 or 72 used in study? to express our gratitude to you and the peer reviewers for the evaluation of our submission entitledTransrenal Mycobacterium tuberculosis DNA in pulmonary tuberculosis patients during the first 14 days of treatmentFollowing the helpful comments provided we have revised the manuscript accordingly.
Reviewer #2: I have a couple of comments questions: What about the actual Ct values when was the lowest Ct value observed?In lines 168-68 the authors state they were not able to compare Ct values.How consistent were the Ct values between individual patients, what was the range of Ct values obtained for all patinets?